Endy:STN Chemostat Protocol
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Set up o/n culture in supM9 media in a small flask at 37 C.
- Take OD600 of o/n culture. Dilute back in the morning into 25 mL/chamber supM9 (~1:100)
- When culture reaches ~OD 2.0 to add 20 mL to each chamber.
- Run at 37 C. The bubbler should be set to the white dot for 6 chambers (med/high) and the effluent needle should be set to around the 24 mL mark to maintain a volume of 20 mL.
- Add the media input (teal, 23-G1 needle) and turn on pump (currently 10 rpm for 20 mL/chamber). After about 1 hour, check that the culture volume is 20 mL and adjust effluent as necessary.
- Run with bleach solution, then H20 after use. Autoclave all components on fast (22 psi, 250 F, 30 min.).
- This water bath should be filled with dI. Top up frequently with dI kept at 37 C.
- Use 1_5_05 supplemented M9 minimal media with AMP (50 ug/mL) or Kan (20 ug/mL) as needed.
- Measure the flow rate by the effluent. 10 rpm gives about 0.25 mL/min, which is about right, but flow rate should be checked during each run.
- Mark 18 mL, 20 mL, and 22 mL on chemostat chambers to assist in monitoring volume.
- It is best to inoculate the chemostat in the early afternoon in order that OD600 may be monitored over the first several hours. The cultures should be at steady-state in the morning (day 2). OD600 should be monitored throughout the day (~every 2 hrs) and samples drawn for steady-state measurements in the afternoon.